This application relates to a peptide-conjugated oligonucleotide therapeutic agent and to methods of using same in therapy.
Sequence-specific antisense oligonucleotides are well known both as a concept for providing targeted therapy, and as myriad specific agents directed to specific targets. Sequence-specific antisense oligonucleotides interact with mRNA having a complementary sequence to interfere with expression of the protein encoded by the mRNA. Crooke, S. T. “Basic Principles of Antisense Therapeutics” in Antisense Research and Application, Pages 1-52, Springer (1998). Because the sequence of the antisense can be selected to complement the mRNA for just one protein, sequence-specific antisense oligonucleotides have been seen as offering vast potential for the treatment of many types of disease characterized by protein over-expression. To date, however, this potential has not been realized, owing in part to the inability to reliably deliver effective amounts of the oligonucleotide into the cells containing the target mRNA in vivo.
Various strategies have been proposed to overcome these difficulties so that antisense therapy can realize its potential. In some proposals, vectors are introduced which lead to the in situ production of antisense species (either full length or as oligonucleotides) using cellular mechanisms for the production of polynucleotides. See Weiss et al., Cell. Mol. Life Sci. 1999, 55:334-358. Others have suggested coupling the oligonucleotide to various moieties to enhance delivery of the oligonucleotide to cells. See Manoharan, M., Antisense and Nucleic Acid Drug Development. 2002, 12(2): 103-128
Peptides that specifically home to tumor endothelial cells in various tissues are known. For example, Porkka et al. Proc. Nat'l Acad. Sci. (USA) 99: 7444-7449 (2002), which is incorporated herein by reference, describe peptides that home to the nuclei of tumor cells and tumor endothelial cells in vivo. Other cell-type specific homing peptides are described in US Patent Publication Nos. US2003/0152578, US2003/0149235, and US2002/0041898, which are incorporated herein by reference. US Patent Publication No. 2003/0152578 relates to conjugates of these homing peptides with drugs or with detectable labels. US Patent Publication No. 2002/0041898 mentions conjugates of homing peptides with various types of molecules including antisense oligonucleotides, but does not disclose any specific method for this conjugation. U.S. Pat. No. 5,670,617 discloses conjugation of nucleic acids with the HIV tat protein as a transport protein to enhance intracellular delivery.
Harrison et al., Nucleic Acids Res. (1998) 26(13): 3136-3145 discloses synthesis of peptide-oligonucleotide conjugates using 4-(maleimidomethyl)-1-cyclohexane carboxylic acid N-hydroxysuccinimde ester (SMCC) as a linker. This paper reports only the use of short peptides, however, to allow assessment of the affect of peptide sequence on antisense activity. These approaches have not provided a general answer to the issue delivery of antisense to cells of interest for efficient reduction of expression of a target protein. When a targeting moiety is added to an oligonucleotide, it may limit the ability of that oligonucleotide to interact with target mRNA for example as a consequence of steric or chemical (hydrophilic/hydrophobic) interactions, alter the ability of the molecule to pass through the cell membrane and/or change the intracellular trafficking pattern for the molecule such that it sequestered in or excreted from the cell, or otherwise made less available for interaction with target nucleotides in the cell.